Peptide thioesters are important compounds in protein chemistry and are widely used in peptide synthesis.
As peptide synthesis, solid-phase peptide synthesis has been known previously. In solid-phase peptide synthesis, however, reaction products cannot be obtained at high purity unless each condensation reaction proceeds to almost 100%, and about 20 to 50 residues are thus a limit for application to solid-phase peptide synthesis.
To synthesize a peptide with 100 or more residues, native chemical ligation (NCL) using peptide thioesters has been developed. The technique of this method includes, for example, the following two steps. In the first step, a first peptide having a thioester moiety at its C-terminus and a second peptide having cysteine at its N-terminus are intramolecularly reacted chemoselectively, and an S-acyl isopeptide intermediate is produced by a thiol-exchange reaction (S—S acyl transfer) in which thiol (SH group) is selectively reacted with carbonyl carbon of a thioester. Next, in the second step, the intermediate produced in the first step spontaneously undergoes an intramolecular S—N acyl transfer reaction to give a native amide bond at the ligation site while regenerating the cysteine side-chain thiol. In this method, even when compounds such as peptides having many functional groups are reacted, the C-terminus of one peptide is selectively ligated to the N-terminus of the other peptide. Moreover, according to this method, two peptide chains are ligated via a peptide bond by simply mixing unprotected starting material peptides in a buffer solution. Furthermore, this method requires no deprotection step because the peptide obtained by this method is free from protecting groups. This remarkably simplified method has been widely used as a first choice peptide synthesis (Non-patent Literature 1).
However, how to produce the above peptide thioester currently poses a problem.
A generally known method for producing a peptide thioester comprises a solid-phase synthesis involving a thioester bond. This method, however, has a defect because the thioester bond is decomposed by the base treatment in the deprotection step, and the C-terminal amino acid is easily racemized (Non-patent Literature 1).
In particular, there has been no simple methodology for chemically synthesizing a peptide thioester from a peptide having a native sequence.
Under the current situation described above, the development of a novel method for producing a peptide thioester based on chemical synthesis techniques has been desired.
Further, C-terminal amidated peptide amides are important for the expression of physiological functions and the stability of the peptide itself, and many of them have by now been placed on the market as peptide pharmaceuticals.
Thus, the development of a novel method for producing a peptide amide based on chemical synthesis or direct genetic engineering technique has been drawing attention.